Effect of ARTEMIS (DCLRE1C) deficiency and microinjection timing on editing efficiency during somatic cell nuclear transfer and in vitro fertilization using the CRISPR/Cas9 system.
Effect of ARTEMIS (DCLRE1C) deficiency and microinjection timing on editing efficiency during somatic cell nuclear transfer and in vitro fertilization using the CRISPR/Cas9 system.
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DOI：
10.1016/j.theriogenology.2021.04.003
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发表时间：
2021-08
期刊：
影响因子：
2.8
通讯作者：
Ross JW
中科院分区：
文献类型：
--
作者：
Li Y;Adur MK;Wang W;Schultz RB;Hale B;Wierson W;Charley SE;McGrail M;Essner J;Tuggle CK;Ross JW
利用CRISPR/Cas9系统，有效地向哺乳动物基因组引入位点特异性遗传修饰的能力得到了极大的提高。CRISPR/Cas9是一种强大的工具，用于通过在DNA中引起双链断裂（DSBs）来产生遗传修饰。Artemis （ART，也称为DCLRE1C）是一种核蛋白，是通过典型的非同源末端连接（c-NHEJ）途径在DNA修复中连接DSB末端所必需的。在这项工作中，我们测试了ART缺乏是否会影响CRISPR/Cas9诱导体细胞dsb后的DNA修复。我们还利用猪体外受精（IVF）和体细胞核移植（SCNT）衍生的胚胎证明了显微注射时间对胚胎发育能力和CRISPR/Cas9系统破坏白细胞介素2受体亚单位γ (IL2RG)位点的基因靶向效率的影响。与未注射对照相比，在受精后4小时和8小时注射CRISPR/Cas9的体外受精受精卵对卵裂和囊胚率没有影响。基因修饰率较高，受精后4 h注射的囊胚为53.3%(9/16)，而受精后8 h注射的囊胚为11.1%（1/9）。SCNT来源的胚胎在化学激活后8小时进行显微注射，与未注射的对照组相比，囊胚发育率降低，但基因修饰效率更高，为66.7%，而激活后4小时注射组的基因修饰效率为25%。此外，我们观察到雄性ART - / -和ART+/ -猪胎儿成纤维细胞（pFF）细胞的修饰率（分别为2.5%和1.9%）低于ART完整细胞系（8.3%）。有趣的是，女性ART - / -和ART+/ - pFF细胞的修饰率（分别为4.2%和10.1%）与ART完整细胞相似。本研究证明了显微注射时间和ART缺乏等多种参数对体外源性猪胚胎基因编辑效率的复杂影响。
The ability to efficiently introduce site-specific genetic modifications to the mammalian genome has been dramatically improved with the use of the CRISPR/Cas9 system. CRISPR/Cas9 is a powerful tool used to generate genetic modifications by causing double-strand breaks (DSBs) in DNA. Artemis (ART; also known as DCLRE1C), is a nuclear protein and is essential for DSB end joining in DNA repair via the canonical non-homologous end joining (c-NHEJ) pathway. In this work, we tested whether ART deficiency affects DNA repair following CRISPR/Cas9 induced DSBs in somatic cells. We also demonstrated the effect of microinjection timing on embryo developmental ability and gene targeting efficiency of CRISPR/Cas9 system to disrupt the interleukin 2 receptor subunit gamma (IL2RG) locus using porcine in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryos. In comparison to non-injected controls, CRISPR/Cas9 injection of IVF derived zygotes at 4 h and 8 h after fertilization did not impact cleavage and blastocyst rate. Gene modification rate was observed to be higher, 53.3% (9/16) in blastocysts injected 4 h post-fertilization as compared to 11.1% (1/9) in blastocysts injected 8 h post-fertilization. Microinjection 8 h after chemical activation of SCNT derived embryos decreased blastocyst development rate compared to non-injected controls but showed a higher gene modification efficiency of 66.7% as compared to 25% in the 4 h post-activation injection group. Furthermore, we observed that male ART−/− and ART+/− porcine fetal fibroblast (pFF) cells showed lower modification rates (2.5% and 1.9%, respectively) as compared to the ART intact cell line (8.3%). Interestingly, the female ART−/− and ART+/− pFF cells had modification rates (4.2% and 10.1%, respectively) similar to those seen in the ART intact cells. This study demonstrates the complex effect of various parameters such as microinjection timing and ART deficiency on gene editing efficiency in in vitro derived porcine embryos.
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作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
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